Diverse function of aromatase and the N-terminal sequence deleted form

Abstract

The diverse function of human placental aromatase including estradiol 6α-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6α-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6α,7α-3H2,4-14C]estradiol, 7-ethoxycoumarin, and [N-methyl-3H3]cocaine. 6α-Hydroxy[7α-3H,4-14C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6α-3H label was established. The initial rate kinetics of the 6α-hydroxylation gave Km of 4.3 μM, Vmax of 4.02 nmol min−1mg−1, and turnover rate of 0.27 min−1. Testosterone competed dose-dependently with the 6α-hydroxylation and showed the Ki of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 μM, Vmax of 12.5 nmol min−1mg−1 and turnover rate of 1.06 min−1. The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 μM, Vmax of 4.76 nmol min−1mg−1, and turnover rate of 0.49 min−1. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min−1mg−1 (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min−1.